Conus gamma-carboxylase

ABSTRACT

The present invention is relates to a γ-carboxylase from Conus snails, a nucleic acid sequence encoding the Conus γ-carboxylase and to a method for using the nucleic acid or protein sequences for preparing γ-carboxylated proteins.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application is a continuation of U.S. patentapplication Ser. No. 10/213,439 filed 7 Aug. 2002. The presentapplication is related to and claims priority under 35 U.S.C. §119(e) toU.S. provisional patent application Ser. No. 60/310,496 filed 8 Aug.2001, incorporated herein by reference.

[0002] This invention was made with Government support under Grant No.PO1 GM48677 awarded by the National Institute of General MedicalSciences, National Institutes of Health, Bethesda, Md. The United StatesGovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] The present invention relates to a γ-carboxylase from Conussnails, a nucleic acid sequence encoding the Conus γ-carboxylase and toa method for using the nucleic acid or protein sequences for preparingγ-carboxylated proteins.

[0004] The publications and other materials used herein to illuminatethe background of the invention, and in particular, cases to provideadditional details respecting the practice, are incorporated byreference, and for convenience are referenced in the following text bynumber and are listed numerically in the appended Bibliography.

[0005] The vitamin K-dependent γ-carboxylation of glutamate residues wasoriginally discovered as a novel post-translational modification in theblood coagulation cascade (Stenflo et al., 1974); some of the keyclotting factors such as prothrombin must be γ-carboxylated in order forproper blood clotting to occur. Somewhat later, this post-translationalmodification was also found in certain bone proteins (Price andWilliamson, 1985). In mammalian blood coagulation and bone Gla proteins,γ-carboxylation of glutamate residues is carried out by a vitaminK-dependent carboxylase. A conserved motif (Price et al., 1987)γ-carboxylation recognition sequence in the propeptide sequence bindsthe γ-carboxylase and is required for a polypeptide substrate to be ahigh affinity target for the γ-carboxylase.

[0006] This modification was restricted to these rather specializedmammalian systems until a very unusual peptide, conantokin-G, wasdescribed from the venom of the predatory marine snail, Conus geographus(McIntosh et al., 1984). Conantokin-G is a 17-amino acid peptide thatinhibits the N-methyl- D-aspartate receptor (Olivera et al., 1990).Unlike most Conus peptides, which are multiply disulfide-bonded,conantokin-G has no disulfide cross-links but has five residues ofγ-carboxyglutamate residues; this remains the highest density ofγ-carboxyglutamate found in any functional gene product characterized todate. Most of the biologically active components of the Conus venom aremultiply disulfide bonded peptides (the conotoxins). These have beenshown to be initially translated as prepropeptide precursors, which arethen post-translationally processed to yield the maturedisulfide-crosslinked conotoxin. Conantokin-G differs strikingly frommost conotoxins not only in having γ-carboxyglutamate residues, but alsobecause it has no disulfide crosslinks. U.S. Pat. No. 6,197,535describes the analysis of the conantokin-G precursor and sequencerecognition by a γ-carboxylase for the maturation of the functionalconantokin-G peptide. It was found a γ-carboxylation recognitionsequence is included in the −1 to −20 region of the conantokin-Gprepropeptide. This sequence appears to increase the affinity of theConus carboxylase by approximately two orders of magnitude.

[0007] The presence of γ-carboxyglutamate in a non-mammalian system wasinitially controversial because vitamin K-dependent carboxylation ofglutamate residues had primarily been thought to be a highly specializedmammalian innovation. However, conantokin-G is only one member of afamily of peptides; a variety of other conantokins have been foundincluding conantokin-T and conantokin-R from two other fish-hunting conesnails (Haack et al., 1990; White et al., 1997). All three peptides havea high content of γ-carboxyglutamate (4-5 residues). γ-Glutamylcarboxylase has been purified from mammalian sources (Wu et al., 1991a;Berkner et al., 1992), has been expressed both in mammalian and insectcell lines (Wu et al., 1991b; Roth et al., 1993) and has been purifiedfrom Conus (U.S. Pat. No. 6,197,535). Recently it was shown that, as isthe case in the mammalian system, the carboxylation reaction in Conusvenom ducts absolutely requires vitamin K, and the net carboxylationincreases greatly in the presence of high concentrations of ammoniumsulfate. In these respects, the mammalian and the Conus γ-carboxylationvenom systems are very similar (Stanley et al., 1997).

[0008] Knobloch and Suttie (1987) and Cheung et al. (1989) found thatthe propeptide sequences of Factors IX and X at micromolarconcentrations stimulated the carboxylation of oligopeptide substrates,suggesting a probable positive allosteric effector role. In addition,the propeptide at micromolar concentrations acted as a competitiveinhibitor of carboxylation of a substrate whose sequences were based onresidues −18 to +10 of prothrombin (Ulrich et al., 1988). Similarly, theConus propeptide (−20 to −1) inhibits the carboxylation ofpropeptide-containing substrates, (e.g., −10.Pro-E.Con-G and−20.Pro-E.Con-G) (U.S. Pat. No. 6,197,535).

[0009] The orientation in which a Glu presents itself to the active siteof the carboxylase may determine whether it will be carboxylated. In thecase of Con-G not all the Glu residues are γ-carboxylated (e.g., Glu² isnot carboxylated, whereas Glu³ and Glu⁴ are carboxylated). The solutionstructures of Con-G and Con-T as determined by CD and NMR spectroscopy(Skjaebaek et al., 1997; Warder et al., 1997) are a mixture of α and 3₁₀helices. Rigby et al. (1997) also determined the structure of themetal-free conformer of conantokin-G by NMR spectroscopy. In all ofthese structures, the Gla residues are on the same side of theconantokin structure; this would allow a membrane-bound enzyme to carryout efficient carboxylation of Glu residues oriented in the samedirection with optimum stereochemistry.

[0010] There is a need in the art to identify the nucleic acid sequenceencoding Conus γ-carboxylase, to identify the sequence of Conusγ-carboxylase, and to use the nucleic acids or proteins in theproduction of γ-carboxylated proteins.

SUMMARY OF THE INVENTION

[0011] The present invention is relates to a γ-carboxylase from Conussnails, a nucleic acid sequence encoding the Conus γ-carboxylase and toa method for using the nucleic acid or protein sequences for preparingγ-carboxylated proteins.

[0012] Thus, one aspect of the invention is directed to the amino acidsequence of C. textile γ-carboxylase. The amino acid sequence of C.textile γ-carboxylase is set forth in SEQ ID NO:2 or SEQ ID NO:4.

[0013] A second aspect of the invention is directed to a nucleic acidencoding a C. textile γ-carboxylase. A preferred nucleotide sequence ofthe nucleic acid is set forth in SEQ ID NO:1 or SEQ ID NO:3.

[0014] A third aspect of the invention is directed to amino acidsequences and nucleic acid sequences of other Conus γ-carboxylases, aswell as amino acid sequences and nucleic acid sequences having 95%identity with the disclosed sequences.

[0015] A fourth aspect of the invention is directed to vectorscontaining the γ-carboxylase encoding nucleic acid.

[0016] A fifth aspect of the invention is directed to host cellscontaining an expression cassette with the γ-carboxylase encodingnucleic acid.

[0017] A sixth aspect of the invention is directed to host cellscontaining an expression cassette with the γ-carboxylase encodingnucleic acid sequence and an expression cassette with a nucleic acidsequence encoding a protein which is γ-carboxylated. Such proteinsinclude conantokins and other vitamin K-dependent proteins.

[0018] A seventh aspect of the invention is directed to the use of aγ-carboxylase for the preparation of γ-carboxylated proteins (the termused herein to refer to proteins which are γ-carboxylated), such asconantokins and other vitamin K-dependent proteins.

[0019] An eighth aspect of the invention is directed to the use of aγ-carboxylase nucleic acid for the preparation of γ-carboxylatedproteins, such as conantokins and other vitamin K-dependent proteins.

DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention is relates to a γ-carboxylase from Conussnails, a nucleic acid sequence encoding the Conus γ-carboxylase and toa method for using the nucleic acid or protein sequences for preparingγ-carboxylated proteins.

[0021] In one aspect, the present invention relates to the amino acidsequence of C. textile γ-carboxylase. The amino acid sequence of C.textile γ-carboxylase is set forth in SEQ ID NO:2 or SEQ ID NO:4. In afurther embodiment, the present invention relates to a γ-carboxylasewhich has at least 95% identity with the amino acid sequence set forthin SEQ ID NO:2 or SEQ ID NO:4 and which has γ-carboxylation activity.The γ-carboxylation activity can be assayed as described herein toidentify those proteins having the proper biological activity.

[0022] In a second aspect, the present invention relates to a nucleicacid encoding a C. textile γ-carboxylase. A preferred nucleic acidsequence is set forth in SEQ ID NO:1 or SEQ ID NO:3. In a furtherembodiment, the present invention relates to a γ-carboxylase encodingnucleic acid which has at least 95% identity with the nucleotidesequence set forth in SEQ ID NO:1 or SEQ ID NO:3. The encodedγ-carboxylase has γ-carboxylation activity which can be assayed asdescribed herein to identify those nucleic acids which encode proteinshaving the proper biological activity.

[0023] In a third aspect, the present invention relates to vectorscontaining the nucleic acid encoding a γ-carboxylase of the presentinvention. In one embodiment, the vector is an expression vector.

[0024] In a fourth aspect, the present invention relates to host cellscontaining an expression cassette or expression vector with theγ-carboxylase encoding nucleic acid of the present invention. The hostcells produce the γ-carboxylase when grown under suitable growthconditions.

[0025] In a fifth aspect, the present invention relates to host cellscontaining an expression cassette or expression vector with theγ-carboxylase encoding nucleic acid of the present invention and anexpression cassette with a nucleic acid sequence encoding a proteinwhich is γ-carboxylated. Such proteins include conantokins and othervitamin K-dependent proteins.

[0026] In a sixth aspect, the present invention relates to the use of aγ-carboxylase of the present invention for the preparation ofγ-carboxylated proteins, such as conantokins and other vitaminK-dependent proteins.

[0027] In a seventh aspect, the present invention relates to the use ofa γ-carboxylase encoding nucleic acid of the present invention for thepreparation of γ-carboxylated proteins, such as conantokins and othervitamin K-dependent proteins.

[0028] A nucleic acid or fragment thereof has substantial identity withanother if, when optimally aligned (with appropriate nucleotideinsertions or deletions) with the other nucleic acid (or itscomplementary strand), there is nucleotide sequence identity in at leastabout 60% of the nucleotide bases, usually at least about 70%, moreusually at least about 80%, preferably at least about 90%, and morepreferably at least about 95-98% of the nucleotide bases. A protein orfragment thereof has substantial identity with another if, optimallyaligned, there is an amino acid sequence identity of at least about 30%identity with an entire naturally-occurring protein or a portionthereof, usually at least about 70% identity, more usually at leastabout 80% identity, preferably at least about 90% identity, and morepreferably at least about 95-98% identity.

[0029] Identity means the degree of sequence relatedness between twopolypeptide or two polynucleotides sequences as determined by theidentity of the match between two strings of such sequences, such as thefull and complete sequence. Identity can be readily calculated. Whilethere exist a number of methods to measure identity between twopolynucleotide or polypeptide sequences, the term “identity” is wellknown to skilled artisans (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). Methods commonly employed to determine identity betweentwo sequences include, but are not limited to those disclosed in Guideto Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego,1994, and Carillo, H., and Lipman, D., SIAM J Applied Math. 48: 1073(1988). Preferred methods to determine identity are designed to give thelargest match between the two sequences tested. Such methods arecodified in computer programs. Preferred computer program methods todetermine identity between two sequences include, but are not limitedto, GCG (Genetics Computer Group, Madison Wis.) program package(Devereux, J., et al., Nucleic Acids Research 12(1). 387 (1984)),BLASTP, BLASTN, FASTA (Altschul et al. (1990); Altschul et al. (1997)).The well-known Smith Waterman algorithm may also be used to determineidentity.

[0030] As an illustration, by a polynucleotide having a nucleotidesequence having at least, for example, 95% “identity” to a referencenucleotide sequence of is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence. In other words, toobtain a polynucleotide having a nucleotide sequence at least 95%identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. These mutations of the reference sequence may occur at the 5or 3 terminal positions of the reference nucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongnucleotides in the reference sequence or in one or more contiguousgroups within the reference sequence.

[0031] Alternatively, substantial homology or (similarity) exists when anucleic acid or fragment thereof will hybridize to another nucleic acid(or a complementary strand thereof) under selective hybridizationconditions, to a strand, or to its complement. Selectivity ofhybridization exists when hybridization which is substantially moreselective than total lack of specificity occurs. Typically, selectivehybridization will occur when there is at least about 55% homology overa stretch of at least about 14 nucleotides, preferably at least about65%, more preferably at least about 75%, and most preferably at leastabout 90%. The length of homology comparison, as described, maybe overlonger stretches, and in certain embodiments will often be over astretch of at least about nine nucleotides, usually at least about 20nucleotides, more usually at least about 24 nucleotides, typically atleast about 28 nucleotides, more typically at least about 32nucleotides, and preferably at least about 36 or more nucleotides.

[0032] Nucleic acid hybridization will be affected by such conditions assalt concentration, temperature, or organic solvents, in addition to thebase composition, length of the complementary strands, and the number ofnucleotide base mismatches between the hybridizing nucleic acids, aswill be readily appreciated by those skilled in the art. Stringenttemperature conditions will generally include temperatures in excess of30° C., typically in excess of 37° C., and preferably in excess of 45°C. Stringent salt conditions will ordinarily be less than 1000 mM,typically less than 500 mM, and preferably less than 200 mM. However,the combination of parameters is much more important than the measure ofany single parameter. The stringency conditions are dependent on thelength of the nucleic acid and the base composition of the nucleic acid,and can be determined by techniques well known in the art. See, e.g.,Asubel, 1992; Wetmur and Davidson, 1968.

[0033] Thus, as herein used, the term “stringent conditions” meanshybridization will occur only if there is at least 95% and preferably atleast 97% identity between the sequences. Such hybridization techniquesare well known to those of skill in the art. Stringent hybridizationconditions are as defined above or, alternatively, conditions underovernight incubation at 42° C. in a solution comprising: 50% formamide,5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate(pH7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/mldenatured, sheared salmon sperm DNA, followed by washing the filters in0.1×SSC at about 65° C.

[0034] The terms “isolated”, “substantially pure”, and “substantiallyhomogeneous” are used interchangeably to describe a protein orpolypeptide which has been separated from components which accompany itin its natural state. A monomeric protein is substantially pure when atleast about 60 to 75% of a sample exhibits a single polypeptidesequence. A substantially pure protein will typically comprise about 60to 90% W/W of a protein sample, more usually about 95%, and preferablywill be over about 99% pure. Protein purity or homogeneity may beindicated by a number of means well known in the art, such aspolyacrylamide gel electrophoresis of a protein sample, followed byvisualizing a single polypeptide band upon staining the gel. For certainpurposes, higher resolution may be provided by using HPLC or other meanswell known in the art which are utilized for purification.

[0035] Large amounts of the nucleic acids of the present invention maybe produced by (a) replication in a suitable host or transgenic animalor (b) chemical synthesis using techniques well known in the art.Nucleic acids made by either of these techniques are also referred to assynthetic nucleic acids herein. Constructs prepared for introductioninto a prokaryotic or eukaryotic host may comprise a replication systemrecognized by the host, including the intended polynucleotide fragmentencoding the desired polypeptide, and will preferably also includetranscription and translational initiation regulatory sequences operablylinked to the polypeptide encoding segment. Such vectors may be preparedby means of standard recombinant techniques well known in the art. Seefor example, see Ausbel (1992); Sambrook and Russell (2001); and U.S.Pat. No. 5,837,492.

[0036] Large amounts of the protein of the present invention may beproduced by (a) expression in a suitable host or transgenic animal or(b) chemical synthesis using techniquest well known in the art. Proteinsacids made by either of these techniques are also referred to assynthetic proteins herein. Expression vectors may include, for example,an origin of replication or autonomously replicating sequence (ARS) andexpression control sequences, a promoter, an enhancer and necessaryprocessing information sites, such as ribosome-binding sites, RNA splicesites, polyadenylation sites, transcriptional terminator sequences, andMRNA stabilizing sequences. Secretion signals may also be included whereappropriate which allow the protein to cross and/or lodge in cellmembranes, and thus attain its functional topology, or be secreted fromthe cell. Such vectors may be prepared by means of standard recombinanttechniques well known in the art. See for example, see Ausbel (1992);Sambrook and Russell (2001); and U.S. Pat. No. 5,837,492.

[0037] The γ-carboxylase of the present invention is isolated followingexpression in a suitable host or chemical synthesis using techniqueswell known in the art. The isolated γ-carboxylase of the presentinvention is used to γ-carboxylate γ-carboxylated proteins, such asconantokins and other vitamin K-dependent proteins. The γ-carboxylase iscontacted with the pro-protein which contains the γ-carboxylationrecognition sequence and allowed to γ-carboxylate the protein. Theγ-carboxylated protein is isolated and purified using techniques wellknown in the art.

[0038] The nucleic acid encoding γ-carboxylase of the present inventionis used to γ-carboxylate γ-carboxylated proteins, such as conantokinsand other vitamin K-dependent proteins, in vivo using techniques wellknown in the art. In one embodiment, a suitable host is prepared whichcontains an expression vector containing a γ-carboxylase encodingnucleic acid of the present invention and an expression vectorcontaining a nucleic acid encoding a γ-carboxylated protein, suchconantokin and other vitamin K-dependent protein. Nucleic acids encodingconantokins are well known in the art. See U.S. Pat. No. 6,172,041.Nucleic acids encoding other vitamin K-dependent proteins are also wellknown in the art. In a second embodiment, a suitable host is preparedwhich contains an expression vector containing a γ-carboxylase encodingnucleic acid and a nucleic acid encoding a γ-carboxylated protein, suchconantokin and other vitamin K-dependent protein. In either embodiment,the host cells are grown under conditions suitable for growth andexpression of the γ-carboxylase and the γ-carboxylated protein. Theγ-carboxylase acts on the γ-carboxylated protein in vivo to properlyγ-carboxylate the Glu residues in the protein.

EXAMPLES

[0039] The present invention is described by reference to the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques specifically described below were utilized.

Example 1 In Vitro γ-Carboxylation of ConG with γ-Carboxylase

[0040] In order to ascertain the fidelity of γ-carboxylation, it isessential to determine if the appropriate Glu residues are beingmodified and if the modification goes to completion. ConG, −20pro-ConGand pro-ConG are used as substrates to determine fidelity ofcarboxylation in vitro with the Conus γ-carboxylase produced inaccordance with the present invention as described in U.S. Pat. No.6,197,535. The identification of Gla by routine amino acid sequencing isnot efficient due to poor recovery of Gla residues. In the presentmodified method, Gla-containing peptides are decarboxylated by heatingunder vacuum. The Gla residues are converted to Glu which can then besequenced. If ¹⁴CO₂ is incorporated in the γ-carboxylation reaction,half of the molecules in the decarboxylated product will contain ¹⁴CO₂covalently linked to the γ-C of modified Glu residues. Thus, bymonitoring the radioactivity recovered at each step of the sequencingreaction, the position of Gla residues can be determined.

[0041] Products of the γ-carboxylation reaction are purified using aWaters Oasis

HLB Extraction Cartridge followed by reversed phase HPLC using Vydac C₁₈column. The [¹⁴C]-containing fractions are dried and sequencedchemically with concomitant determination of radioactivity at eachposition in the sequence. When −20.pro-ConG and pro-ConG are used as thesubstrate, the radioactivity-containing fraction from the reversed phaseHPLC column are dried and digested with endoproteinase Lys C. This isdone to reduce the length of the peptide for sequencing withoutinterfering with the identification of the γ-carboxylated residues. TheLys C digest is purified using a Waters Oasis

HLB Extraction Cartridge followed by reversed phase HPLC using Vydac C₁₈column. The radioactivity eluted as a single peak coincidental with theA₂₂₀ peak. The chemical sequence of the material had the expectedsequence.

[0042] The Gla determinations are carried out on a mixture of unmodifiedand variously modified substrate molecules. On the basis of theseexperiments, it is not possible to assign Gla residues to individualpost-translationally modified substrate molecules. However, an averagepicture emerged. As in the case of the native product, Glu² is notcarboxylated. The rest of the Glu residues are carboxylated.

Example 2 Cloning of Conus Textile γ-Carboxylase cDNA

[0043] The full-length C. textile γ-carboxylase cDNA was isolated byreverse transcription-PCR (RT-PCR) of venom duct RNA, using primersdesigned from conserved regions of mammalian γ-carboxylase proteins. Anumber of different internal primer sets were utilized to generateoverlapping segments of the C. textile γ-carboxylase sequence. Most ofthe internal region of the C. textile cDNA was obtained in this manner,generating sequence to within ˜100 amino acids of the putative N- andC-termini of the protein. To obtain the ends of the cDNA sequence,nested PCR primers based on the C. textile cDNA sequence were used in 5′and 3′ RACE to identify the transcription start site and poly Atermination site, respectively. Merging of these overlapping segmentsgenerated a ˜2460 bp cDNA sequence with a single long open reading frameencoding a protein of 799 amino acids. This C. textile protein hassubstantial homology to the mammalian γ-carboxylase. Overall homology ofthe Conus sequence to the mammalian enzymes is ˜50%, although distinctregions within the protein show substantially higher and lower levels ofsequence conservation. The RT-PCR with degenerate primers consistentlyidentified the sequence presented here, with no evidence for any otherrelated γ-carboxylase isoform being expressed in the C. textile venomduct. Much of the degenerate PCR used to initially clone the C. textilesequence employed non-proofreading thermostable polymerases and manycycles of PCR, both conditions that could introduce minor sequenceerrors. To obtain an accurate sequence, primers designed to the verymost 5′ and 3′ ends of the C. textile cDNA sequence were used to amplifythe full-length 2460 bp cDNA using a proofreading thermostablepolymerase mixture and a minimal number of PCR cycles. This Long,Accurate-PCR generated the predicted 2460 bp product in good yield, withno evidence for alternative-splice isoforms of the C. textileγ-carboxylase mRNA being expressed in the venom duct. The full-lengthcDNA product was cloned and completely sequenced on both strands,yielding the sequence presented here.

[0044] The C. textile γ-carboxylase cDNA has a short 5′ untranslatedregion of ˜50 bp, and the first ATG start codon encountered initiatesthe long open reading frame encoding the γ-carboxylase protein. The cDNAsequence obtained by 3′ RACE terminates in a poly A tail, and this ispreceded by a typical poly A addition signal (AATAA). An unusual featureis that the open reading frame lacks a typical stop codon (TAG, TAG,TGA) and instead continues into the poly A tail. We considered thepossibility that the 3′ RACE technique had not identified the true 3′end of the mRNA, or that a sequencing error obscured a stop codon thatis present, but various experiments tend to rule this out. A variety of3′ RACE experiments have been performed, using different PCR primers andcDNA preparations with different 3′ adapters, yet all of theseexperiments consistently identify the same 3′ poly A site. Numerousdifferent PCR product clones representing this 3′ region have beenthoroughly sequenced, and there are no ambiguities on the sequencingchromatograms that would suggest a sequencing anomaly that could changethe open reading frame.

[0045] Finally, 3′ RACE was used to isolate the corresponding region ofγ-carboxylase cDNA from the venom duct RNA of two other Conus species,omaria and episcopatus, that are snail-hunting species related totextile. The 3′ RACE identified poly A sites at essentially the samelocation in all three different species. The DNA sequences (andcorresponding protein sequence) were highly homologous between the threespecies, but there is sequence variation as expected between species.Even though the sequence varies between the species, in all three theopen reading frame lacks a typical stop codon and extends into the polyA tail. This suggests that our initial finding was not just a cloning orsequence artifact restricted to C. textile, but is a conserved featureof the γ-carboxylase mRNA across related Conus species. It is possiblethat Conus recognizes an atypical triplet as a termination codon, orthat post-translation processing generates the true C-terminus of theConus γ-carboxylase enzyme. Assuming that it terminates at the poly Atail, the size of the Conus γ-carboxylase protein that we haveidentified (799 amino acids) is very similar to that of the mammalianγ-carboxylase enzymes, so the overall size of the proteins appears to beconserved.

[0046] The nucleic acid sequence (SEQ ID NO:1 or 3) and amino acidsequence (SEQ ID NO:2 or 4) for C. textile γ-carboxylase are set forthin Tables 1 and 2, respectively. The 3′ nucleic acid sequence (SEQ IDNO:5 or 7) and C-terminal amino acid sequence (SEQ ID NO:6 or 8) for C.omaria are set forth in Tables 3 and 4, respectively. The 3′ nucleicacid sequence (SEQ ID NO:9 or 11) and C-terminal amino acid sequence(SEQ ID NO:10 or 12) for C. episcopatus are set forth in Tables 5 and 6,respectively.

[0047] The sequences for C. omaria and C. episcopatus represent about200-220 amino acids at the C-terminus, starting at a positioncorresponding to approximately 590 of C. textile. Both of thesesequences have several base pair and amino acid changes compared to theC. textile sequence as is expected for species homologs, although thesequence identity is quite high. All three species have the poly A tailin roughly the same location (the C. textile sequence is about 30 bplonger) and none of the three species has a typical stop codon. TABLE 1Nucleic Acid Sequence of C. textile γ-CarboxylaseATCTTTGTGAGCGTGATCCATCGCACAAACCATGCAAAGGCCAGGCAAGAAAGTGGCTGCTGATTCAGAG(SEQ ID NO: 1)GAATCAAATGACATCAGCCAACAAGCAGAAAACAGAGACCAGCTCCTCCCCCAGGAAGCCAGTCCCAAAGCGTGTGAGGAAGAGGACACAGAGGATGAAGAGGAAGAAGAGGACAAGTTCTACAAACTCTTTGGTTTCAGCTTGAGCGACCTCAAGTCATGGGACAGCTTTGTTCGTCTGTTGTCGCGCCCCGCTGACCCTGCCGGTCTGGCTTATATCCGTGTCACTTATGGGTTTTTGATGATGTGGGACGTGTTTGAGGAAAGGGGCCTGTCCCGTGCAGATATGCGATGGGGTGATGATGAGGCATGCAGGTTTCCTCTCTTCGACTTCATGCAACCCTTGCCCCTGCACATGATGGTCCTGCTGTACCTGATCATGCTGATTGGAACAGGAGGAATTCTATTAGGAGCCAAGTACCGTGTGTGCTGCGTTATGCACCTGCTGCCCTACTGGTACATAGTGCTTCTGGACGAGTGCAGTTGGAACAATCACTCCTATCTGTTTGGTCTCCTCTCTTTCCTCCTTCTGCTTTGCGATGCTAACCACTACTGGTCCATGGACGGTCTGTTCAATGCCAAGGTTCGAAATACCGATGTTCCCTTGTGGAACTACACCCTCCTACGTACACAGGTGTTTCTGGTGTACTTTTTGGCTGGGCTGAAGAAACTGGACATGGACTGGATCGCTGGTTACTCCATGGGCCCTTTGAGTGATCATTGGGTCTTTTACCCGTTTACGTTCCTGATGACAGAAGACCAGGTGAGTGTGCTTGTGGTCCACCTGGGTGGACTTGCCATTGACTTGTTCGTCGGCTACCTGCTCTTCTTTGACAAGACACCACCGATCGGTGTCATTATCAGTTCGTCATTCCACCTGATGAATGCACAGATGTTCAGCATAGGAATGTTTCCCTATGCCATGTTGGGTTTGACGCCTGTGTTCTTCTATGCCAACTGGCCGAGGGCCCCGTTTCGCCGCATTCCACGATCCTTGAGGATTCTTACCCCTGATGATGGAGAGGATGATACGCTGCCTTCGGAGAAGTGCTTATACACAAAAGAACAGGCCAAACCAGAACTGGCCAGCACCCCTGAGCATGAAAACACTGCAGTCCGCAAACAGTTGACACCACCCACTCAGCCCACGTTCCGGCATCATGCTGCCGCTGCCTTCACCGTTTTCTTCATTCTGTGGCAGATGTTTTTGCCTTTCTCTCATTTTATCACAAAGGGCAACAACAGCTGGACCCAGGGACTCTACGGCTACTCCTGGGACATGATGGTTCACACCCGCAGCACTCAGCACACCAGGATCTCCTTCATCAACAAGGACACAGGAGAGCGAGGGTTCCTGGACCCGCAGGCATGGAGCAAGTCACATCGATGGGCGCATAACGCTAAGATGATGAAGCAGTACGCCAGGTGCATCGCTCGCCGACTGAAGAAGCATGAAATCGACAATGTGGAAATCTATTTTGATGTCTGGATATCTCTGAATCATCGCTTCCAGCAACGGATCGTGAACCCCAATGTGGACATTTTAACAGCCGAATGGAGTGTCTTTAAGTCCACTCCATGGATGATGCCCTTGCTGGTCGACTTGTCTAATTGGCGAAGCAAGTTGAAAGAGATTGAGGACGACATTTTCAACTCAACCGACCTGTATGAAATAGTCTTTCTGGCTGACTTTCCTGGTTTGTACCTGGAGAACTTTGTCCACGGCAGCGTCGGGAGTCTCAACATCTCTGTACTGCAGGGCCAGGTGGTGGTGGAGGTGCTTCCAGAGGAGGACAGTCTAGAAGAGCCCTACAACATCAGCATCAGTGATGGCCAAGAGTCATTGATTCCCACAGGGGTGTTCCACAAGGTGTACACAGTGTCTGAAGTGCCCTCCTGTTACATGTACATCTACATGGTCACGGAAGAGACAGAGTTCCTTGAGAAACTCAAAGAGCTGGAACACGCCCTCAACGGCTCCCTGGATGCTCCAGTTCCAGACAAGTTTGCCGAAGATCCTAAACTTGATCAGTATATGGAGGTACTCAAAACGAAGAATGCAACTCCACCACCAACCTCTCAAGAGGAGCAAAGTTTCATACAGCTGTTTATGAGTTTTCTGAAAATGCATTATATGTCTATGTATCGTGGACTGCAGCTGATAAAAGGCGCCATGTGGTCCATGTACTCCGGGGAATCTTACCGAGAGTTCTTGAAGAAACTGGAGCTACAGAAAATGCTGGCGGAGAATGCCACCCTGGTGGCAAACGCCACCCAAGGGGTGAATAACACCCAGACGATGAACAACACCTTGAACAACACCAAGGAGAAAGACAACACCCAAAGGGTTAACAAACCGATGCAAAGGCCAGGCAAGAAAGTGGCTGCTGATTCAGAGGAATCAAATGACATCAGCCAACAAGCAGAAA(SEQ ID NO: 3)ACAGAGACCAGCTCCTCCCCCAGGAAGCCAGTCCCAAAGCGTGTGAGGAAGAGGACACAGAGGATGAAGAGGAAGAAGAGGACAAGTTCTACAAACTCTTTGGTTTCAGCTTGAGCGACCTCAAGTCATGGGACAGCTTTGTTCGTCTGTTGTCGCGCCCCGCTGACCCTGCCGGTCTGGCTTATATCCGTGTCACTTATGGGTTTTTGATGATGTGGGACGTGTTTGAGGAAAGGGGCCTGTCCCGTGCAGATATGCGATGGGGTGATGATGAGGCATGCAGGTTTCCTCTCTTCGACTTCATGCAACCCTTGCCCCTGCACATGATGGTCCTGCTGTACCTGATCATGCTGATTGGAACAGGAGGAATTCTATTAGGAGCCAAGTACCGTGTGTGCTGCGTTATGCACCTGCTGCCCTACTGGTACATAGTGCTTCTGGACGAGTGCAGTTGGAACAATCACTCCTATCTGTTTGGTCTCCTCTCTTTCCTCCTTCTGCTTTGCGATGCTAACCACTACTGGTCCATGGACGGTCTGTTCAATGCCAAGGTTCGAAATACGGATGTTCCCTTGTGGAACTACACCCTCCTACGTACACAGGTGTTTCTGGTGTACTTTTTGGCTGGGCTGAAGAAACTGGACATGGACTGGATCGCTGGTTACTCCATGGGCCGTTTGAGTGATCATTGGGTCTTTTACCCGTTTACGTTCCTGATGACAGAAGACCAGGTGAGTGTGCTTGTGGTCCACCTGGGTGGACTTGCCATTGACTTGTTCGTGGGCTACCTGCTCTTCTTTGACAAGACACGACCGATCGGTGTCATTATCAGTTCGTCATTCCACCTGATGAATGCACAGATGTTCAGCATAGGAATGTTTCCGTATGCCATGTTGGGTTTGACGCCTGTGTTCTTCTATGCCAACTGGCCGAGGGCCCTGTTTCGCCGCATTCCACGATCCTTGAGGATTCTTACCCCTGATGATGGAGAGGATGATACGCTGCCTTCGGAGAAGTGCTTATACACAAAAGAACAGGCCAAACCAGAACTGGCCAGCACCCCTGAGCATGAAAACACTGCAGTCCGCAAACAGTTGACACCACCCACTCAGCCCACGTTCCGGCATCATGCTGCCGCTGCCTTCACCGTTTTCTTCATTCTGTGGCAGATGTTTTTGCCTTTCTCTCATTTTATCACAAAGGGCAACAACAGCTGGACCCAGGGACTCTACGGCTACTCCTGGGACATGATGGTTCACACCCGCAGCACTCAGCACACCAGGATCTCCTTCATCAACAAGGACACAGGAGAGCGAGGGTTCCTGGACCCGCAGGCATGGAGCAAGTCACATCGATGGGCGCATAACGCTAAGATGATGAAGCAGTACGCCAGGTGCATCGCTCGCCGACTGAAGAAGCATGAAATCGACAATGTGGAAATCTATTTTGATGTCTGGATATCTCTGAATCATCGCTTCCAGCAACGGATCGTGAACCCCAATGTGGACATTTTAACAGCCGAATGGAGTGTCTTTAAGTCCACTCCATGGATGATCCCCTTGCTGGTCGACTTGTCTAATTGGCGAAGCAAGTTGAAAGAGATTGAGGACGACATTTTCAACTCAACCGACCTGTATGAAATAGTCTTTCTGGCTGACTTTCCTGGTTTGTACCTGGAGAACTTTGTCCACGGCAGCGTCGGGAGTCTCAACATCTCTGTACTGCAGGGCCAGGTGGTGGTGGAGGTGCTTCCAGAGGAGGACAGTCTAGAAGAGCCCTACAACATCAGCATCAGTGATGGCCAAGAGTCATTGATTCCCACAGGGGTGTTCCACAAGGTGTACACAGTGTCTGAAGTGCCCTCCTGTTACATGTACATCTACATGGTCACGGAAGAGACAGAGTTCCTTGAGAAACTCAAAGAGCTGGAACACGCCCTCAACGGCTCCCTGGATGCTCCAGTTCCAGACAAGTTTGCCGAAGATCCTAAACTTGATCAGTATATGGAGGTACTCAAAACGAAGAATGCAACTCCACCACCAACCTCTCAAGAGGAGCAAAGTTTCATACAGCTGTTTATGAGTTTTCTGAAAATGCATTATATGTCTATGTATCGTGGACTGCAGCTGATAAAAGGCGCCATGTGGTCCATGTACTCCGGGGAATCTTACCGAGAGTTCTTGAAGAAACTGGAGCTACAGAAAATGCTGGCGGAGAATGCCACCCTGGTGGCAAACGCCACCCAAGGGGTGAATAACACCCAGACGATGAACAACACCTTGAACAACACCAAGGAGAAAGACAACACCCAAAGGGTTAACAAACCGCAGGAAAAGAAGGCCCCCCAGAAGGCAGACAGCCCCTAACAGCATCTCTGCAGAATGAGGGCGTCATGCCTCTGTTCTGCATTGTAAATCTTCAATGTCAGACTCGCTGTCATGAGTCAGGATGCCAAGGGTTGATTCTAAATGAAAAAA

[0048] TABLE 2 Protein Sequence of C. textile γ-CarboxylaseMQRPGKKVAADSEESNDISQQAENRDQLLPQEASPKACEEEDTEDEEEEEDKFYKLFGFSLSDLKSWDSF(SEQ ID NO: 2)VRLLSRPADPAGLAYIRVTYGFLMMWDVFEERGLSRADMRWGDDEACRFPLFDFMQPLPLHMMVLLYLIMLIGTGGILLGAKYRVCCVMHLLPYWYIVLLDECSWNNHSYLFGLLSFLLLLCDANHYWSMDGLFNAKVRNTDVPLWNYTLLRTQVFLVYFLAGLKKLDMDWIAGYSMGRLSDHWVFYPFTFLMTEDQVSVLVVHLGGLAIDLFVGYLLFFDKTPPIGVIISSSFHLMNAQMFSIGMFPYAMLGLTPVFFYANWPRAPFRRIPRSLRILTPDDGEDDTLPSEKCLYTKEQAKPELASTPEHENTAVRKQLTPPTQPTFRHHAAAAFTVFFILWQMFLPFSHFITKGNNSWTQGLYGYSWDMMVHTRSTQHTRISFINKDTGERGFLDPQAWSKSHRWAHNAKMMKQYARCIARRLKKHEIDNVEIYFDVWISLNHRFQQRIVNPNVDILTAEWSVFKSTPWMMPLLVDLSNWRSKLKEIEDDIFNSTDLYEIVFLADFPGLYLENFVHGSVGSLNISVLQGQVVVEVLPEEDSLEEPYNISISDGQESLIPTGVFHKVYTVSEVPSCYMYIYMVTEETEFLEKLKELEHALNGSLDAPVPDKFAEDPKLDQYMEVLKTKNATPPPTSQEEQSFIQLFMSFLKMHYMSMYRGLQLIKGAMWSMYSGESYREFLKKLELQKMLAENATLVANATQGVNNTQTMNNTLNNTKEKDNTQRVNKPMQRPGKKVAADSEESNDISQQAENRDQLLPQEASPKACEEEDTEDEEEEEDKFYKLFGFSLSDLKSWDSF(SEQ ID NO: 4)VRLLSRPADPAGLAYIRVTYGFLMMWDVFEERGLSRADMRWGDDEACRFPLFDFMQPLPLHMMVLLYLIMLIGTGGILLGAKYRVCCVMHLLPYWYIVLLDECSWNNHSYLFGLLSFLLLLCDANHYWSMDGLFNAKVRNTDVPLWNYTLLRTQVFLVYFLAGLKKLDMDWIAGYSMGRLSDHWVFYPFTFLMTEDQVSVLVVHLGGLAIDLFVGYLLFFDKTRPIGVIISSSFHLMNAQMFSIGMFPYAMLGLTPVFFYANWPRALFRRIPRSLRILTPDDGEDDTLPSEKCLYTKEQAKPELASTPEHENTAVRKQLTPPTQPTFRHHAAAAFTVFFILWQMFLPFSHFITKGNNSWTQGLYGYSWDMMVHTRSTQHTRISFINKDTGERGFLDPQAWSKSHRWAHNAKMMKQYARCIARRLKKHEIDNVEIYFDVWISLNHRFQQRIVNPNVDILTAEWSVFKSTPWMMPLLVDLSNWRSKLKEIEDDIFNSTDLYEIVFLADFPGLYLENFVHGSVGSLNISVLQGQVVVEVLPEEDSLEEPYNISISDGQESLIPTGVFHKVYTVSEVPSCYMYIYMVTEETEFLEKLKELEHALNGSLDAPVPDKFAEDPKLDQYMEVLKTKNATPPPTSQEEQSFIQLFMSFLKMHYMSMYRGLQLIKGAMWSMYSGESYREFLKKLELQKMLAENATLVANATQGVNNTQTMNNTLNNTKEKDNTQRVNKPQEKKAPQKADSP

[0049] TABLE 3 3′ Nucleic Acid Sequence of C. omaria γ-CarboxylaseGGGAGTCTCAACATCTCTGTACTGCAGGGGCAGGTGGTGGTGGAGGTGCTTCCAGAGGAGGACAGTCTAG(SEQ ID NO: 5)AAAAACCCTACAACATCAGCATCAATGATGGCCACGAGTCATTGATTCCCACAGGGGTATTCCACAAGGTGTACACAGTGTCTGAAGTGCCCTCCTGTTACATGTACATCTACATGGTCACGGAAGAGACGGAGTTCTTTGAGAACGTCAAAGAGCTGGAACACGCCCTCAACGGCTCCCTGGATGCTCCAGTTCCAGACAAGTTTGCCAAAGATCCTAAACTTGATCAATATATGGAGCTACTCAAAGTGAAGAATGCAGCTCCACCACCGGCCCCTCGAGCGGAGAGAAGTTTCATAGAGCTGTTTATGAGTTTTCTGAAAATGCATTATATGTCTATGTATCGTGGACTGCAGCTGATAAAAGGCGCCGTGTGGTCCATGTACTCTGGGGAATCTTACCGAGAGTACCTGAAGGAACTGGAATTACAGGCAATGCTGGGGGAGAATGCCACCCTGGTGGCAAACGCCACCCAAGGGGTGAATAACACCCAGACGATGAACAACACCTTATTGAACAACACCAAAAAAAAAAAAAAAAAAGGGAGTCTCAACATCTCTGTACTGCAGGGCCAGGTGGTGGTGGAGGTGCTTCCAGAGGAGGACAGTCTAG(SEQ ID NO: 7)AAAAACCCTACAACATCAGCATCAATGATGGCCACGAGTCATTGATTCCCACAGGGGTATTCCACAAGGTGTACACAGTGTCTGAAGTGCCCTCCTGTTACATGTACATCTACATGGTCACGGAAGAGACGGAGTTCTTTGAGAACGTCAAAGAGCTGGAACACGCCCTCAACGGCTCCCTGGATGCTCCAGTTCCAGACAAGTTTGCCAAAGATCCTAAACTTGATCAATATATGGAGGTACTCAAAGTGAAGAATGCAGCTCCACCACCGGCCCCTCGAGCGGAGAGAAGTTTCATAGAGCTGTTTATGAGTTTTCTGAAAATGCATTATATGTCTATGTATCGTGGACTGCAGCTGATAAAAGGCGCCGTGTGGTCCATGTACTCTGGGGAATCTTACCGAGAGTACCTGAAGGAACTGGAATTACAGGCAATGCTGGGGGAGAATGCCACCCTGGTGGCAAACGCCACCCAAGGGGTGAATAACACCCAGACGATGAACAACACCTTATTGAACAACACCAAGGAGAAAAACAACACCCAAAGGGTTAACAAGCCGCAGGAAAAGAAGGCCCCCCAGAAGGCAGACAGCCCCTAACAGCATCTCTGCAGAATGAGGGCATCATGCCTCTGTTCTGCATTTTAAATCTTCGATGTCAGACACGCTGTCATGAGTCAGGATGCCAAGGGTTGATTCTAAATGAAAAAA

[0050] TABLE 4 C-Terminal Protein Sequence of C. omaria γ-CarboxylaseGSLNISVLQGQVVVEVLPEEDSLEKPYNISINDGHESLIPTGVFHKVYTVSEVPSCYMYIYMVTEETEFF(SEQ ID NO: 6)ENVKELEHALNGSLDAPVPDKFAKDPKLDQYMEVLKVKNAAPPPAPRAERSFIELFMSFLKMHYMSMYRGLQLIKGAVWSMYSGESYREYLKELELQAMLGENATLVANATQGVNNTQTMNNTLLNNTKKKKKKGSLNISVLQGQVVVEVLPEEDSLEKPYNISINDGHESLIPTGVFHKVYTVSEVPSCYMYIYMVTEETEFF(SEQ ID NO: 8)ENVKELEHALNGSLDAPVPDKFAKDPKLDQYMEVLKVKNAAPPPAPRAERSFIELFMSFLKMHYMSMYRGLQLIKGAVWSMYSGESYREYLKELELQAMLGENATLVANATQGVNNTQTMNNTLLNNTKEKNNTQRVNKPQEKKAPQKADSP

[0051] TABLE 5 3′ Nucleic Acid Sequence of C. episcopatus γ-CarboxylaseGGGAGTCTCAACATCTCTGTACTGCAGGGCCAGGTGGTGGTGGAGGTGCTTCCAGAGGAGGACAGTCTAG(SEQ ID NO: 9)AACAGCCCTACAACATCAGCATCAGTGATGGCCACGAGTCATTGATTCCCACAGGGGTGTTCCACAAGGTGTACACAGTGTCTGAAGTGCCCTCCTGTTACATGTACATCTAGATGGTCACGGAAGAGACGGAGTTCTTTGAGAACCTCAAAGAGCTGGAACACGCCCTCAACGGCTCCCTGGATGCTCCAGTACCAGACAAGTTTGCCAAAGATCCTAAACTTGATCAATATATGGAGGTACTCAAGGTGAAGAATGCAGCTCCACCACCGGCCCCTCCAGCGGACAGAAGTTTCATACAGCTGTTTATGAGTTTTCTGAAAATGCATTATATGTCTATGTATCGTGGACTGCAGCTGATAAAAGGCGCCGTGTGGTCCATGTACTCTGGGGAATCTTACCGAGAGTACCTGAAGGAACTGGAGCTACAGGCAATGCTGGGGGAGAATGTCACCCTGGTGGCAAATGCCACCGAAGGGGTGAATAAAACCCAGATGATGAACAACACCTTGAACAACACCAAAAAAAAAAAAAAAAAAGGGAGTCTCAACATCTCTGTACTGCAGGGCCAGGTGGTGGTGGAGGTGCTTCCAGAGGAGGACAGTCTAG(SEQ ID NO: 11)AACAGCCCTACAACATCAGCATCAGTGATGGCCACGAGTCATTGATTCCCACAGGGGTGTTCCACAAGGTGTACACAGTGTCTGAAGTGCCCTCCTGTTACATGTACATCTACATGGTCACGGAAGAGACGGAGTTCTTTGAGAACCTCAAAGAGCTGGAACACGCCCTCAACGGCTCCCTGGATGCTCCAGTACCAGACAAGTTTGCCAAAGATCCTAAACTTGATCAATATATGGAGGTACTCAAAGTGAAGAATGCAGCTCCACCACCGGCCCCTCCAGCGGACAGAAGTTTCATACAGCTGTTTATGAGTTTTCTGAAAATGCATTATATGTCTATGTATCGTGGACTGCAGCTGATAAAAGGCGCCGTGTGGTCCATGTACTCTGGGGAATCTTACCGAGAGTACCTGAAGGAACTGGAGCTACAGGCAATGCTGGGGGAGAATGTCACCCTGGTGGCAAATGCCACCCAAGGGGTGAATAAAACCCAGATGATGAACAACACCTTGAACAACACCAAGGAGAAAAACAACACCCAAAGGGTTAACAAGCCGCAGGAAAAGAAGGCCCCCCAGAAGGCAGACAGCCCCTAACAGCATCTCTGCAGAATGAGGGCATCATGCCTCTGTTCTGCATTTTAAATCTTCAATGTCAGACACGCTGTCATGAGTCAGGATGCCAAGGGTTGATTCTAAATGAAAAAA

[0052] TABLE 6 C-Terminal Protein Sequence of C. episcopatusγ-CarboxylaseGSLNISVLQGQVVVEVLPEEDSLEQPYNISISDGHESLIPTGVFHKVYTVSEVPSCYMYIYMVTEETEFF(SEQ ID NO: 10)ENLKELEHALNGSLDAPVPDKFAKDPKLDQYMEVLKVKNAAPPPAPPADRSFIQLFMSFLKMHYMSMYRGLQLIKGAVWSMYSGESYREYLKELELQAMLGENVTLVANATQGVNKTQMMNNTLNNTKKKKKKGSLNISVLQGQVVVEVLPEEDSLEQPYNISISDGHESLIPTGVFHKVYTVSEVPSCYMYIYMVTEETEFF(SEQ ID NO: 12)ENLKELEHALNGSLDAPVPDKFAKDPKLDQYMEVLKVKNAAPPPAPPADRSFIQLFMSFLKMHYMSMYRGLQLIKGAVWSMYSGESYREYLKELELQAMLGENVTLVANATQGVNKTQMMNNTLNNTKEKNNTQRVNKPQEKKAPQKADSP

Example 3 Expression of γ-Carboxylase in Host Cells

[0053] The C. textile γ-carboxylase cDNA sequence is cloned andexpressed as described by Walker et al. (2001). Briefly, the cDNA codingsequence is cloned in frame with green fluorescent protein (GFP) in theexpression plasmid pRmHa-3.GFP (Walker et al., 2001). Expression in thisplasmid is under control of the Drosophila inducible metallothioneinpromoter and carries the alcohol dehydrogenase poly (A) addition signal.Drosophila Schneider 2 (S2) cells are transfected with the resultantplasmid containing the Conus γ-carboxylase coding sequence usingCelIFECTIN™ (Life Technologies). Twenty-four hours after transfection,cells are induced with 0.7 mM CuSO₄. Forty-eight hours aftertransfection, cells are found to express GFP as seen by fluorescentmicroscopy. The plasmid containing the Conus γ-carboxylase codingsequence and the GFP sequence is modified to add a stop codon at the endof the Conus γ-carboxylase coding sequence and to delete the GFP codingsequence.

[0054] This modified expression vector and a vector DNA expressing thehygromyocin gene are used to cotransfect Drosophila S2 cells.Hygromyocin resistant cells are selected and individual clones areexpanded. The expanded clones are analyzed for expression of Conusγ-carboxylase. Briefly, the cells are induced with 0.7 mM CuSO₄ andharvested 48 hours after induction. Cells are washed twice withphosphate-buffered saline and resuspended in buffer containing 25 MM4-morpholinepropanesulfonic acid, Ph7.0, 0.5 M NaCl, 0.2%3-[(3-chloramidopropyl)dimethyl-ammonio]-1-propane sulfonicacid/poshphatidyl choline, 2 MM EDTA, 2 MM dithiothreitol, 0.2 μg/mlleupeptin, 0.8 μg/ml pepstatin and 0.04 Mg/ml phenylmethylsulfonylfluoride. The cell suspension is briefly sonicated and incubated in icefor 20 min. The lysate is assayed for Conus γ-carboxylase activity asdescribed in Example 1. The isolated Conus γ-carboxylase is found to bebiologically active and to properly γ-carboxylate ConG, i.e. Glu² is notγ-carboxylated while the remaining Glu residues are γ-carboxylated. Thecells expressing the Conus γ-carboxylase are grown and maintained.

Example 4 Synthesis of γ-Carboxylated ConG in Host Cells

[0055] The cDNA sequence coding for the ConG propeptide (U.S. Pat. No.6,172,041) is cloned and expressed as described by Walker et al. (2001).Briefly, the cDNA for the ConG propeptide sequence is cloned intopRmHa-3.GFP under control of the Drosophila metallothionenin promoter asdescribed in Example 3. The resultant plasmid is modified to insert astop codon and to delete the GFP coding sequence as described in Example3. This expression vector is used to transfect cells expressingγ-carboxylase prepared in Example 3. Cells expressing γ-carboxylase andConG propeptide are selected and expanded. ConG is isolated from thesecells and analyzed for proper γ-carboxylation as described in Example 1.The Glu residues in ConG are found to be properly γ-carboxylated, i.e.Glu² is not γ-carboxylated while the remaining Glu residues areγ-carboxylated.

[0056] It will be appreciated that the methods and compositions of theinstant invention can be incorporated in the form of a variety ofembodiments, only a few of which are disclosed herein. It will beapparent to the artisan that other embodiments exist and do not departfrom the spirit of the invention. Thus, the described embodiments areillustrative and should not be construed as restrictive.

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[0079] U.S. Pat. No. 6,172,041

[0080] U.S. Pat. No. 6,197,535

1 12 1 2428 DNA Conus textile misc_feature (32)..(34) start codon 1atctttgtga gcgtgatcca tcgcacaaac catgcaaagg ccaggcaaga aagtggctgc 60tgattcagag gaatcaaatg acatcagcca acaagcagaa aacagagacc agctcctccc 120ccaggaagcc agtcccaaag cgtgtgagga agaggacaca gaggatgaag aggaagaaga 180ggacaagttc tacaaactct ttggtttcag cttgagcgac ctcaagtcat gggacagctt 240tgttcgtctg ttgtcgcgcc ccgctgaccc tgccggtctg gcttatatcc gtgtcactta 300tgggtttttg atgatgtggg acgtgtttga ggaaaggggc ctgtcccgtg cagatatgcg 360atggggtgat gatgaggcat gcaggtttcc tctcttcgac ttcatgcaac ccttgcccct 420gcacatgatg gtcctgctgt acctgatcat gctgattgga acaggaggaa ttctattagg 480agccaagtac cgtgtgtgct gcgttatgca cctgctgccc tactggtaca tagtgcttct 540ggacgagtgc agttggaaca atcactccta tctgtttggt ctcctctctt tcctccttct 600gctttgcgat gctaaccact actggtccat ggacggtctg ttcaatgcca aggttcgaaa 660tacggatgtt cccttgtgga actacaccct cctacgtaca caggtgtttc tggtgtactt 720tttggctggg ctgaagaaac tggacatgga ctggatcgct ggttactcca tgggccgttt 780gagtgatcat tgggtctttt acccgtttac gttcctgatg acagaagacc aggtgagtgt 840gcttgtggtc cacctgggtg gacttgccat tgacttgttc gtgggctacc tgctcttctt 900tgacaagaca ccaccgatcg gtgtcattat cagttcgtca ttccacctga tgaatgcaca 960gatgttcagc ataggaatgt ttccgtatgc catgttgggt ttgacgcctg tgttcttcta 1020tgccaactgg ccgagggccc cgtttcgccg cattccacga tccttgagga ttcttacccc 1080tgatgatgga gaggatgata cgctgccttc ggagaagtgc ttatacacaa aagaacaggc 1140caaaccagaa ctggccagca cccctgagca tgaaaacact gcagtccgca aacagttgac 1200accacccact cagcccacgt tccggcatca tgctgccgct gccttcaccg ttttcttcat 1260tctgtggcag atgtttttgc ctttctctca ttttatcaca aagggcaaca acagctggac 1320ccagggactc tacggctact cctgggacat gatggttcac acccgcagca ctcagcacac 1380caggatctcc ttcatcaaca aggacacagg agagcgaggg ttcctggacc cgcaggcatg 1440gagcaagtca catcgatggg cgcataacgc taagatgatg aagcagtacg ccaggtgcat 1500cgctcgccga ctgaagaagc atgaaatcga caatgtggaa atctattttg atgtctggat 1560atctctgaat catcgcttcc agcaacggat cgtgaacccc aatgtggaca ttttaacagc 1620cgaatggagt gtctttaagt ccactccatg gatgatgccc ttgctggtcg acttgtctaa 1680ttggcgaagc aagttgaaag agattgagga cgacattttc aactcaaccg acctgtatga 1740aatagtcttt ctggctgact ttcctggttt gtacctggag aactttgtcc acggcagcgt 1800cgggagtctc aacatctctg tactgcaggg ccaggtggtg gtggaggtgc ttccagagga 1860ggacagtcta gaagagccct acaacatcag catcagtgat ggccaagagt cattgattcc 1920cacaggggtg ttccacaagg tgtacacagt gtctgaagtg ccctcctgtt acatgtacat 1980ctacatggtc acggaagaga cagagttcct tgagaaactc aaagagctgg aacacgccct 2040caacggctcc ctggatgctc cagttccaga caagtttgcc gaagatccta aacttgatca 2100gtatatggag gtactcaaaa cgaagaatgc aactccacca ccaacctctc aagaggagca 2160aagtttcata cagctgttta tgagttttct gaaaatgcat tatatgtcta tgtatcgtgg 2220actgcagctg ataaaaggcg ccatgtggtc catgtactcc ggggaatctt accgagagtt 2280cttgaagaaa ctggagctac agaaaatgct ggcggagaat gccaccctgg tggcaaacgc 2340cacccaaggg gtgaataaca cccagacgat gaacaacacc ttgaacaaca ccaaggagaa 2400agacaacacc caaagggtta acaaaccg 2428 2 799 PRT Conus textile 2 Met GlnArg Pro Gly Lys Lys Val Ala Ala Asp Ser Glu Glu Ser Asn 1 5 10 15 AspIle Ser Gln Gln Ala Glu Asn Arg Asp Gln Leu Leu Pro Gln Glu 20 25 30 AlaSer Pro Lys Ala Cys Glu Glu Glu Asp Thr Glu Asp Glu Glu Glu 35 40 45 GluGlu Asp Lys Phe Tyr Lys Leu Phe Gly Phe Ser Leu Ser Asp Leu 50 55 60 LysSer Trp Asp Ser Phe Val Arg Leu Leu Ser Arg Pro Ala Asp Pro 65 70 75 80Ala Gly Leu Ala Tyr Ile Arg Val Thr Tyr Gly Phe Leu Met Met Trp 85 90 95Asp Val Phe Glu Glu Arg Gly Leu Ser Arg Ala Asp Met Arg Trp Gly 100 105110 Asp Asp Glu Ala Cys Arg Phe Pro Leu Phe Asp Phe Met Gln Pro Leu 115120 125 Pro Leu His Met Met Val Leu Leu Tyr Leu Ile Met Leu Ile Gly Thr130 135 140 Gly Gly Ile Leu Leu Gly Ala Lys Tyr Arg Val Cys Cys Val MetHis 145 150 155 160 Leu Leu Pro Tyr Trp Tyr Ile Val Leu Leu Asp Glu CysSer Trp Asn 165 170 175 Asn His Ser Tyr Leu Phe Gly Leu Leu Ser Phe LeuLeu Leu Leu Cys 180 185 190 Asp Ala Asn His Tyr Trp Ser Met Asp Gly LeuPhe Asn Ala Lys Val 195 200 205 Arg Asn Thr Asp Val Pro Leu Trp Asn TyrThr Leu Leu Arg Thr Gln 210 215 220 Val Phe Leu Val Tyr Phe Leu Ala GlyLeu Lys Lys Leu Asp Met Asp 225 230 235 240 Trp Ile Ala Gly Tyr Ser MetGly Arg Leu Ser Asp His Trp Val Phe 245 250 255 Tyr Pro Phe Thr Phe LeuMet Thr Glu Asp Gln Val Ser Val Leu Val 260 265 270 Val His Leu Gly GlyLeu Ala Ile Asp Leu Phe Val Gly Tyr Leu Leu 275 280 285 Phe Phe Asp LysThr Pro Pro Ile Gly Val Ile Ile Ser Ser Ser Phe 290 295 300 His Leu MetAsn Ala Gln Met Phe Ser Ile Gly Met Phe Pro Tyr Ala 305 310 315 320 MetLeu Gly Leu Thr Pro Val Phe Phe Tyr Ala Asn Trp Pro Arg Ala 325 330 335Pro Phe Arg Arg Ile Pro Arg Ser Leu Arg Ile Leu Thr Pro Asp Asp 340 345350 Gly Glu Asp Asp Thr Leu Pro Ser Glu Lys Cys Leu Tyr Thr Lys Glu 355360 365 Gln Ala Lys Pro Glu Leu Ala Ser Thr Pro Glu His Glu Asn Thr Ala370 375 380 Val Arg Lys Gln Leu Thr Pro Pro Thr Gln Pro Thr Phe Arg HisHis 385 390 395 400 Ala Ala Ala Ala Phe Thr Val Phe Phe Ile Leu Trp GlnMet Phe Leu 405 410 415 Pro Phe Ser His Phe Ile Thr Lys Gly Asn Asn SerTrp Thr Gln Gly 420 425 430 Leu Tyr Gly Tyr Ser Trp Asp Met Met Val HisThr Arg Ser Thr Gln 435 440 445 His Thr Arg Ile Ser Phe Ile Asn Lys AspThr Gly Glu Arg Gly Phe 450 455 460 Leu Asp Pro Gln Ala Trp Ser Lys SerHis Arg Trp Ala His Asn Ala 465 470 475 480 Lys Met Met Lys Gln Tyr AlaArg Cys Ile Ala Arg Arg Leu Lys Lys 485 490 495 His Glu Ile Asp Asn ValGlu Ile Tyr Phe Asp Val Trp Ile Ser Leu 500 505 510 Asn His Arg Phe GlnGln Arg Ile Val Asn Pro Asn Val Asp Ile Leu 515 520 525 Thr Ala Glu TrpSer Val Phe Lys Ser Thr Pro Trp Met Met Pro Leu 530 535 540 Leu Val AspLeu Ser Asn Trp Arg Ser Lys Leu Lys Glu Ile Glu Asp 545 550 555 560 AspIle Phe Asn Ser Thr Asp Leu Tyr Glu Ile Val Phe Leu Ala Asp 565 570 575Phe Pro Gly Leu Tyr Leu Glu Asn Phe Val His Gly Ser Val Gly Ser 580 585590 Leu Asn Ile Ser Val Leu Gln Gly Gln Val Val Val Glu Val Leu Pro 595600 605 Glu Glu Asp Ser Leu Glu Glu Pro Tyr Asn Ile Ser Ile Ser Asp Gly610 615 620 Gln Glu Ser Leu Ile Pro Thr Gly Val Phe His Lys Val Tyr ThrVal 625 630 635 640 Ser Glu Val Pro Ser Cys Tyr Met Tyr Ile Tyr Met ValThr Glu Glu 645 650 655 Thr Glu Phe Leu Glu Lys Leu Lys Glu Leu Glu HisAla Leu Asn Gly 660 665 670 Ser Leu Asp Ala Pro Val Pro Asp Lys Phe AlaGlu Asp Pro Lys Leu 675 680 685 Asp Gln Tyr Met Glu Val Leu Lys Thr LysAsn Ala Thr Pro Pro Pro 690 695 700 Thr Ser Gln Glu Glu Gln Ser Phe IleGln Leu Phe Met Ser Phe Leu 705 710 715 720 Lys Met His Tyr Met Ser MetTyr Arg Gly Leu Gln Leu Ile Lys Gly 725 730 735 Ala Met Trp Ser Met TyrSer Gly Glu Ser Tyr Arg Glu Phe Leu Lys 740 745 750 Lys Leu Glu Leu GlnLys Met Leu Ala Glu Asn Ala Thr Leu Val Ala 755 760 765 Asn Ala Thr GlnGly Val Asn Asn Thr Gln Thr Met Asn Asn Thr Leu 770 775 780 Asn Asn ThrLys Glu Lys Asp Asn Thr Gln Arg Val Asn Lys Pro 785 790 795 3 2547 DNAConus textile CDS (1)..(2433) 3 atg caa agg cca ggc aag aaa gtg gct gctgat tca gag gaa tca aat 48 Met Gln Arg Pro Gly Lys Lys Val Ala Ala AspSer Glu Glu Ser Asn 1 5 10 15 gac atc agc caa caa gca gaa aac aga gaccag ctc ctc ccc cag gaa 96 Asp Ile Ser Gln Gln Ala Glu Asn Arg Asp GlnLeu Leu Pro Gln Glu 20 25 30 gcc agt ccc aaa gcg tgt gag gaa gag gac acagag gat gaa gag gaa 144 Ala Ser Pro Lys Ala Cys Glu Glu Glu Asp Thr GluAsp Glu Glu Glu 35 40 45 gaa gag gac aag ttc tac aaa ctc ttt ggt ttc agcttg agc gac ctc 192 Glu Glu Asp Lys Phe Tyr Lys Leu Phe Gly Phe Ser LeuSer Asp Leu 50 55 60 aag tca tgg gac agc ttt gtt cgt ctg ttg tcg cgc cccgct gac cct 240 Lys Ser Trp Asp Ser Phe Val Arg Leu Leu Ser Arg Pro AlaAsp Pro 65 70 75 80 gcc ggt ctg gct tat atc cgt gtc act tat ggg ttt ttgatg atg tgg 288 Ala Gly Leu Ala Tyr Ile Arg Val Thr Tyr Gly Phe Leu MetMet Trp 85 90 95 gac gtg ttt gag gaa agg ggc ctg tcc cgt gca gat atg cgatgg ggt 336 Asp Val Phe Glu Glu Arg Gly Leu Ser Arg Ala Asp Met Arg TrpGly 100 105 110 gat gat gag gca tgc agg ttt cct ctc ttc gac ttc atg caaccc ttg 384 Asp Asp Glu Ala Cys Arg Phe Pro Leu Phe Asp Phe Met Gln ProLeu 115 120 125 ccc ctg cac atg atg gtc ctg ctg tac ctg atc atg ctg attgga aca 432 Pro Leu His Met Met Val Leu Leu Tyr Leu Ile Met Leu Ile GlyThr 130 135 140 gga gga att cta tta gga gcc aag tac cgt gtg tgc tgc gttatg cac 480 Gly Gly Ile Leu Leu Gly Ala Lys Tyr Arg Val Cys Cys Val MetHis 145 150 155 160 ctg ctg ccc tac tgg tac ata gtg ctt ctg gac gag tgcagt tgg aac 528 Leu Leu Pro Tyr Trp Tyr Ile Val Leu Leu Asp Glu Cys SerTrp Asn 165 170 175 aat cac tcc tat ctg ttt ggt ctc ctc tct ttc ctc cttctg ctt tgc 576 Asn His Ser Tyr Leu Phe Gly Leu Leu Ser Phe Leu Leu LeuLeu Cys 180 185 190 gat gct aac cac tac tgg tcc atg gac ggt ctg ttc aatgcc aag gtt 624 Asp Ala Asn His Tyr Trp Ser Met Asp Gly Leu Phe Asn AlaLys Val 195 200 205 cga aat acg gat gtt ccc ttg tgg aac tac acc ctc ctacgt aca cag 672 Arg Asn Thr Asp Val Pro Leu Trp Asn Tyr Thr Leu Leu ArgThr Gln 210 215 220 gtg ttt ctg gtg tac ttt ttg gct ggg ctg aag aaa ctggac atg gac 720 Val Phe Leu Val Tyr Phe Leu Ala Gly Leu Lys Lys Leu AspMet Asp 225 230 235 240 tgg atc gct ggt tac tcc atg ggc cgt ttg agt gatcat tgg gtc ttt 768 Trp Ile Ala Gly Tyr Ser Met Gly Arg Leu Ser Asp HisTrp Val Phe 245 250 255 tac ccg ttt acg ttc ctg atg aca gaa gac cag gtgagt gtg ctt gtg 816 Tyr Pro Phe Thr Phe Leu Met Thr Glu Asp Gln Val SerVal Leu Val 260 265 270 gtc cac ctg ggt gga ctt gcc att gac ttg ttc gtgggc tac ctg ctc 864 Val His Leu Gly Gly Leu Ala Ile Asp Leu Phe Val GlyTyr Leu Leu 275 280 285 ttc ttt gac aag aca cga ccg atc ggt gtc att atcagt tcg tca ttc 912 Phe Phe Asp Lys Thr Arg Pro Ile Gly Val Ile Ile SerSer Ser Phe 290 295 300 cac ctg atg aat gca cag atg ttc agc ata gga atgttt ccg tat gcc 960 His Leu Met Asn Ala Gln Met Phe Ser Ile Gly Met PhePro Tyr Ala 305 310 315 320 atg ttg ggt ttg acg cct gtg ttc ttc tat gccaac tgg ccg agg gcc 1008 Met Leu Gly Leu Thr Pro Val Phe Phe Tyr Ala AsnTrp Pro Arg Ala 325 330 335 ctg ttt cgc cgc att cca cga tcc ttg agg attctt acc cct gat gat 1056 Leu Phe Arg Arg Ile Pro Arg Ser Leu Arg Ile LeuThr Pro Asp Asp 340 345 350 gga gag gat gat acg ctg cct tcg gag aag tgctta tac aca aaa gaa 1104 Gly Glu Asp Asp Thr Leu Pro Ser Glu Lys Cys LeuTyr Thr Lys Glu 355 360 365 cag gcc aaa cca gaa ctg gcc agc acc cct gagcat gaa aac act gca 1152 Gln Ala Lys Pro Glu Leu Ala Ser Thr Pro Glu HisGlu Asn Thr Ala 370 375 380 gtc cgc aaa cag ttg aca cca ccc act cag cccacg ttc cgg cat cat 1200 Val Arg Lys Gln Leu Thr Pro Pro Thr Gln Pro ThrPhe Arg His His 385 390 395 400 gct gcc gct gcc ttc acc gtt ttc ttc attctg tgg cag atg ttt ttg 1248 Ala Ala Ala Ala Phe Thr Val Phe Phe Ile LeuTrp Gln Met Phe Leu 405 410 415 cct ttc tct cat ttt atc aca aag ggc aacaac agc tgg acc cag gga 1296 Pro Phe Ser His Phe Ile Thr Lys Gly Asn AsnSer Trp Thr Gln Gly 420 425 430 ctc tac ggc tac tcc tgg gac atg atg gttcac acc cgc agc act cag 1344 Leu Tyr Gly Tyr Ser Trp Asp Met Met Val HisThr Arg Ser Thr Gln 435 440 445 cac acc agg atc tcc ttc atc aac aag gacaca gga gag cga ggg ttc 1392 His Thr Arg Ile Ser Phe Ile Asn Lys Asp ThrGly Glu Arg Gly Phe 450 455 460 ctg gac ccg cag gca tgg agc aag tca catcga tgg gcg cat aac gct 1440 Leu Asp Pro Gln Ala Trp Ser Lys Ser His ArgTrp Ala His Asn Ala 465 470 475 480 aag atg atg aag cag tac gcc agg tgcatc gct cgc cga ctg aag aag 1488 Lys Met Met Lys Gln Tyr Ala Arg Cys IleAla Arg Arg Leu Lys Lys 485 490 495 cat gaa atc gac aat gtg gaa atc tatttt gat gtc tgg ata tct ctg 1536 His Glu Ile Asp Asn Val Glu Ile Tyr PheAsp Val Trp Ile Ser Leu 500 505 510 aat cat cgc ttc cag caa cgg atc gtgaac ccc aat gtg gac att tta 1584 Asn His Arg Phe Gln Gln Arg Ile Val AsnPro Asn Val Asp Ile Leu 515 520 525 aca gcc gaa tgg agt gtc ttt aag tccact cca tgg atg atg ccc ttg 1632 Thr Ala Glu Trp Ser Val Phe Lys Ser ThrPro Trp Met Met Pro Leu 530 535 540 ctg gtc gac ttg tct aat tgg cga agcaag ttg aaa gag att gag gac 1680 Leu Val Asp Leu Ser Asn Trp Arg Ser LysLeu Lys Glu Ile Glu Asp 545 550 555 560 gac att ttc aac tca acc gac ctgtat gaa ata gtc ttt ctg gct gac 1728 Asp Ile Phe Asn Ser Thr Asp Leu TyrGlu Ile Val Phe Leu Ala Asp 565 570 575 ttt cct ggt ttg tac ctg gag aacttt gtc cac ggc agc gtc ggg agt 1776 Phe Pro Gly Leu Tyr Leu Glu Asn PheVal His Gly Ser Val Gly Ser 580 585 590 ctc aac atc tct gta ctg cag ggccag gtg gtg gtg gag gtg ctt cca 1824 Leu Asn Ile Ser Val Leu Gln Gly GlnVal Val Val Glu Val Leu Pro 595 600 605 gag gag gac agt cta gaa gag ccctac aac atc agc atc agt gat ggc 1872 Glu Glu Asp Ser Leu Glu Glu Pro TyrAsn Ile Ser Ile Ser Asp Gly 610 615 620 caa gag tca ttg att ccc aca ggggtg ttc cac aag gtg tac aca gtg 1920 Gln Glu Ser Leu Ile Pro Thr Gly ValPhe His Lys Val Tyr Thr Val 625 630 635 640 tct gaa gtg ccc tcc tgt tacatg tac atc tac atg gtc acg gaa gag 1968 Ser Glu Val Pro Ser Cys Tyr MetTyr Ile Tyr Met Val Thr Glu Glu 645 650 655 aca gag ttc ctt gag aaa ctcaaa gag ctg gaa cac gcc ctc aac ggc 2016 Thr Glu Phe Leu Glu Lys Leu LysGlu Leu Glu His Ala Leu Asn Gly 660 665 670 tcc ctg gat gct cca gtt ccagac aag ttt gcc gaa gat cct aaa ctt 2064 Ser Leu Asp Ala Pro Val Pro AspLys Phe Ala Glu Asp Pro Lys Leu 675 680 685 gat cag tat atg gag gta ctcaaa acg aag aat gca act cca cca cca 2112 Asp Gln Tyr Met Glu Val Leu LysThr Lys Asn Ala Thr Pro Pro Pro 690 695 700 acc tct caa gag gag caa agtttc ata cag ctg ttt atg agt ttt ctg 2160 Thr Ser Gln Glu Glu Gln Ser PheIle Gln Leu Phe Met Ser Phe Leu 705 710 715 720 aaa atg cat tat atg tctatg tat cgt gga ctg cag ctg ata aaa ggc 2208 Lys Met His Tyr Met Ser MetTyr Arg Gly Leu Gln Leu Ile Lys Gly 725 730 735 gcc atg tgg tcc atg tactcc ggg gaa tct tac cga gag ttc ttg aag 2256 Ala Met Trp Ser Met Tyr SerGly Glu Ser Tyr Arg Glu Phe Leu Lys 740 745 750 aaa ctg gag cta cag aaaatg ctg gcg gag aat gcc acc ctg gtg gca 2304 Lys Leu Glu Leu Gln Lys MetLeu Ala Glu Asn Ala Thr Leu Val Ala 755 760 765 aac gcc acc caa ggg gtgaat aac acc cag acg atg aac aac acc ttg 2352 Asn Ala Thr Gln Gly Val AsnAsn Thr Gln Thr Met Asn Asn Thr Leu 770 775 780 aac aac acc aag gag aaagac aac acc caa agg gtt aac aaa ccg cag 2400 Asn Asn Thr Lys Glu Lys AspAsn Thr Gln Arg Val Asn Lys Pro Gln 785 790 795 800 gaa aag aag gcc ccccag aag gca gac agc ccc taacagcatc tctgcagaat 2453 Glu Lys Lys Ala ProGln Lys Ala Asp Ser Pro 805 810 gagggcgtca tgcctctgtt ctgcattgtaaatcttcaat gtcagactcg ctgtcatgag 2513 tcaggatgcc aagggttgat tctaaatgaaaaaa 2547 4 811 PRT Conus textile 4 Met Gln Arg Pro Gly Lys Lys Val AlaAla Asp Ser Glu Glu Ser Asn 1 5 10 15 Asp Ile Ser Gln Gln Ala Glu AsnArg Asp Gln Leu Leu Pro Gln Glu 20 25 30 Ala Ser Pro Lys Ala Cys Glu GluGlu Asp Thr Glu Asp Glu Glu Glu 35 40 45 Glu Glu Asp Lys Phe Tyr Lys LeuPhe Gly Phe Ser Leu Ser Asp Leu 50 55 60 Lys Ser Trp Asp Ser Phe Val ArgLeu Leu Ser Arg Pro Ala Asp Pro 65 70 75 80 Ala Gly Leu Ala Tyr Ile ArgVal Thr Tyr Gly Phe Leu Met Met Trp 85 90 95 Asp Val Phe Glu Glu Arg GlyLeu Ser Arg Ala Asp Met Arg Trp Gly 100 105 110 Asp Asp Glu Ala Cys ArgPhe Pro Leu Phe Asp Phe Met Gln Pro Leu 115 120 125 Pro Leu His Met MetVal Leu Leu Tyr Leu Ile Met Leu Ile Gly Thr 130 135 140 Gly Gly Ile LeuLeu Gly Ala Lys Tyr Arg Val Cys Cys Val Met His 145 150 155 160 Leu LeuPro Tyr Trp Tyr Ile Val Leu Leu Asp Glu Cys Ser Trp Asn 165 170 175 AsnHis Ser Tyr Leu Phe Gly Leu Leu Ser Phe Leu Leu Leu Leu Cys 180 185 190Asp Ala Asn His Tyr Trp Ser Met Asp Gly Leu Phe Asn Ala Lys Val 195 200205 Arg Asn Thr Asp Val Pro Leu Trp Asn Tyr Thr Leu Leu Arg Thr Gln 210215 220 Val Phe Leu Val Tyr Phe Leu Ala Gly Leu Lys Lys Leu Asp Met Asp225 230 235 240 Trp Ile Ala Gly Tyr Ser Met Gly Arg Leu Ser Asp His TrpVal Phe 245 250 255 Tyr Pro Phe Thr Phe Leu Met Thr Glu Asp Gln Val SerVal Leu Val 260 265 270 Val His Leu Gly Gly Leu Ala Ile Asp Leu Phe ValGly Tyr Leu Leu 275 280 285 Phe Phe Asp Lys Thr Arg Pro Ile Gly Val IleIle Ser Ser Ser Phe 290 295 300 His Leu Met Asn Ala Gln Met Phe Ser IleGly Met Phe Pro Tyr Ala 305 310 315 320 Met Leu Gly Leu Thr Pro Val PhePhe Tyr Ala Asn Trp Pro Arg Ala 325 330 335 Leu Phe Arg Arg Ile Pro ArgSer Leu Arg Ile Leu Thr Pro Asp Asp 340 345 350 Gly Glu Asp Asp Thr LeuPro Ser Glu Lys Cys Leu Tyr Thr Lys Glu 355 360 365 Gln Ala Lys Pro GluLeu Ala Ser Thr Pro Glu His Glu Asn Thr Ala 370 375 380 Val Arg Lys GlnLeu Thr Pro Pro Thr Gln Pro Thr Phe Arg His His 385 390 395 400 Ala AlaAla Ala Phe Thr Val Phe Phe Ile Leu Trp Gln Met Phe Leu 405 410 415 ProPhe Ser His Phe Ile Thr Lys Gly Asn Asn Ser Trp Thr Gln Gly 420 425 430Leu Tyr Gly Tyr Ser Trp Asp Met Met Val His Thr Arg Ser Thr Gln 435 440445 His Thr Arg Ile Ser Phe Ile Asn Lys Asp Thr Gly Glu Arg Gly Phe 450455 460 Leu Asp Pro Gln Ala Trp Ser Lys Ser His Arg Trp Ala His Asn Ala465 470 475 480 Lys Met Met Lys Gln Tyr Ala Arg Cys Ile Ala Arg Arg LeuLys Lys 485 490 495 His Glu Ile Asp Asn Val Glu Ile Tyr Phe Asp Val TrpIle Ser Leu 500 505 510 Asn His Arg Phe Gln Gln Arg Ile Val Asn Pro AsnVal Asp Ile Leu 515 520 525 Thr Ala Glu Trp Ser Val Phe Lys Ser Thr ProTrp Met Met Pro Leu 530 535 540 Leu Val Asp Leu Ser Asn Trp Arg Ser LysLeu Lys Glu Ile Glu Asp 545 550 555 560 Asp Ile Phe Asn Ser Thr Asp LeuTyr Glu Ile Val Phe Leu Ala Asp 565 570 575 Phe Pro Gly Leu Tyr Leu GluAsn Phe Val His Gly Ser Val Gly Ser 580 585 590 Leu Asn Ile Ser Val LeuGln Gly Gln Val Val Val Glu Val Leu Pro 595 600 605 Glu Glu Asp Ser LeuGlu Glu Pro Tyr Asn Ile Ser Ile Ser Asp Gly 610 615 620 Gln Glu Ser LeuIle Pro Thr Gly Val Phe His Lys Val Tyr Thr Val 625 630 635 640 Ser GluVal Pro Ser Cys Tyr Met Tyr Ile Tyr Met Val Thr Glu Glu 645 650 655 ThrGlu Phe Leu Glu Lys Leu Lys Glu Leu Glu His Ala Leu Asn Gly 660 665 670Ser Leu Asp Ala Pro Val Pro Asp Lys Phe Ala Glu Asp Pro Lys Leu 675 680685 Asp Gln Tyr Met Glu Val Leu Lys Thr Lys Asn Ala Thr Pro Pro Pro 690695 700 Thr Ser Gln Glu Glu Gln Ser Phe Ile Gln Leu Phe Met Ser Phe Leu705 710 715 720 Lys Met His Tyr Met Ser Met Tyr Arg Gly Leu Gln Leu IleLys Gly 725 730 735 Ala Met Trp Ser Met Tyr Ser Gly Glu Ser Tyr Arg GluPhe Leu Lys 740 745 750 Lys Leu Glu Leu Gln Lys Met Leu Ala Glu Asn AlaThr Leu Val Ala 755 760 765 Asn Ala Thr Gln Gly Val Asn Asn Thr Gln ThrMet Asn Asn Thr Leu 770 775 780 Asn Asn Thr Lys Glu Lys Asp Asn Thr GlnArg Val Asn Lys Pro Gln 785 790 795 800 Glu Lys Lys Ala Pro Gln Lys AlaAsp Ser Pro 805 810 5 612 DNA Conus omaria 5 gggagtctca acatctctgtactgcagggc caggtggtgg tggaggtgct tccagaggag 60 gacagtctag aaaaaccctacaacatcagc atcaatgatg gccacgagtc attgattccc 120 acaggggtat tccacaaggtgtacacagtg tctgaagtgc cctcctgtta catgtacatc 180 tacatggtca cggaagagacggagttcttt gagaacgtca aagagctgga acacgccctc 240 aacggctccc tggatgctccagttccagac aagtttgcca aagatcctaa acttgatcaa 300 tatatggagg tactcaaagtgaagaatgca gctccaccac cggcccctcg agcggagaga 360 agtttcatag agctgtttatgagttttctg aaaatgcatt atatgtctat gtatcgtgga 420 ctgcagctga taaaaggcgccgtgtggtcc atgtactctg gggaatctta ccgagagtac 480 ctgaaggaac tggaattacaggcaatgctg ggggagaatg ccaccctggt ggcaaacgcc 540 acccaagggg tgaataacacccagacgatg aacaacacct tattgaacaa caccaaaaaa 600 aaaaaaaaaa aa 612 6 204PRT Conus omaria 6 Gly Ser Leu Asn Ile Ser Val Leu Gln Gly Gln Val ValVal Glu Val 1 5 10 15 Leu Pro Glu Glu Asp Ser Leu Glu Lys Pro Tyr AsnIle Ser Ile Asn 20 25 30 Asp Gly His Glu Ser Leu Ile Pro Thr Gly Val PheHis Lys Val Tyr 35 40 45 Thr Val Ser Glu Val Pro Ser Cys Tyr Met Tyr IleTyr Met Val Thr 50 55 60 Glu Glu Thr Glu Phe Phe Glu Asn Val Lys Glu LeuGlu His Ala Leu 65 70 75 80 Asn Gly Ser Leu Asp Ala Pro Val Pro Asp LysPhe Ala Lys Asp Pro 85 90 95 Lys Leu Asp Gln Tyr Met Glu Val Leu Lys ValLys Asn Ala Ala Pro 100 105 110 Pro Pro Ala Pro Arg Ala Glu Arg Ser PheIle Glu Leu Phe Met Ser 115 120 125 Phe Leu Lys Met His Tyr Met Ser MetTyr Arg Gly Leu Gln Leu Ile 130 135 140 Lys Gly Ala Val Trp Ser Met TyrSer Gly Glu Ser Tyr Arg Glu Tyr 145 150 155 160 Leu Lys Glu Leu Glu LeuGln Ala Met Leu Gly Glu Asn Ala Thr Leu 165 170 175 Val Ala Asn Ala ThrGln Gly Val Asn Asn Thr Gln Thr Met Asn Asn 180 185 190 Thr Leu Leu AsnAsn Thr Lys Lys Lys Lys Lys Lys 195 200 7 780 DNA Conus omaria CDS(1)..(666) 7 ggg agt ctc aac atc tct gta ctg cag ggc cag gtg gtg gtg gaggtg 48 Gly Ser Leu Asn Ile Ser Val Leu Gln Gly Gln Val Val Val Glu Val 15 10 15 ctt cca gag gag gac agt cta gaa aaa ccc tac aac atc agc atc aat96 Leu Pro Glu Glu Asp Ser Leu Glu Lys Pro Tyr Asn Ile Ser Ile Asn 20 2530 gat ggc cac gag tca ttg att ccc aca ggg gta ttc cac aag gtg tac 144Asp Gly His Glu Ser Leu Ile Pro Thr Gly Val Phe His Lys Val Tyr 35 40 45aca gtg tct gaa gtg ccc tcc tgt tac atg tac atc tac atg gtc acg 192 ThrVal Ser Glu Val Pro Ser Cys Tyr Met Tyr Ile Tyr Met Val Thr 50 55 60 gaagag acg gag ttc ttt gag aac gtc aaa gag ctg gaa cac gcc ctc 240 Glu GluThr Glu Phe Phe Glu Asn Val Lys Glu Leu Glu His Ala Leu 65 70 75 80 aacggc tcc ctg gat gct cca gtt cca gac aag ttt gcc aaa gat cct 288 Asn GlySer Leu Asp Ala Pro Val Pro Asp Lys Phe Ala Lys Asp Pro 85 90 95 aaa cttgat caa tat atg gag gta ctc aaa gtg aag aat gca gct cca 336 Lys Leu AspGln Tyr Met Glu Val Leu Lys Val Lys Asn Ala Ala Pro 100 105 110 cca ccggcc cct cga gcg gag aga agt ttc ata gag ctg ttt atg agt 384 Pro Pro AlaPro Arg Ala Glu Arg Ser Phe Ile Glu Leu Phe Met Ser 115 120 125 ttt ctgaaa atg cat tat atg tct atg tat cgt gga ctg cag ctg ata 432 Phe Leu LysMet His Tyr Met Ser Met Tyr Arg Gly Leu Gln Leu Ile 130 135 140 aaa ggcgcc gtg tgg tcc atg tac tct ggg gaa tct tac cga gag tac 480 Lys Gly AlaVal Trp Ser Met Tyr Ser Gly Glu Ser Tyr Arg Glu Tyr 145 150 155 160 ctgaag gaa ctg gaa tta cag gca atg ctg ggg gag aat gcc acc ctg 528 Leu LysGlu Leu Glu Leu Gln Ala Met Leu Gly Glu Asn Ala Thr Leu 165 170 175 gtggca aac gcc acc caa ggg gtg aat aac acc cag acg atg aac aac 576 Val AlaAsn Ala Thr Gln Gly Val Asn Asn Thr Gln Thr Met Asn Asn 180 185 190 acctta ttg aac aac acc aag gag aaa aac aac acc caa agg gtt aac 624 Thr LeuLeu Asn Asn Thr Lys Glu Lys Asn Asn Thr Gln Arg Val Asn 195 200 205 aagccg cag gaa aag aag gcc ccc cag aag gca gac agc ccc 666 Lys Pro Gln GluLys Lys Ala Pro Gln Lys Ala Asp Ser Pro 210 215 220 taacagcatctctgcagaat gagggcatca tgcctctgtt ctgcatttta aatcttcgat 726 gtcagacacgctgtcatgag tcaggatgcc aagggttgat tctaaatgaa aaaa 780 8 222 PRT Conusomaria 8 Gly Ser Leu Asn Ile Ser Val Leu Gln Gly Gln Val Val Val Glu Val1 5 10 15 Leu Pro Glu Glu Asp Ser Leu Glu Lys Pro Tyr Asn Ile Ser IleAsn 20 25 30 Asp Gly His Glu Ser Leu Ile Pro Thr Gly Val Phe His Lys ValTyr 35 40 45 Thr Val Ser Glu Val Pro Ser Cys Tyr Met Tyr Ile Tyr Met ValThr 50 55 60 Glu Glu Thr Glu Phe Phe Glu Asn Val Lys Glu Leu Glu His AlaLeu 65 70 75 80 Asn Gly Ser Leu Asp Ala Pro Val Pro Asp Lys Phe Ala LysAsp Pro 85 90 95 Lys Leu Asp Gln Tyr Met Glu Val Leu Lys Val Lys Asn AlaAla Pro 100 105 110 Pro Pro Ala Pro Arg Ala Glu Arg Ser Phe Ile Glu LeuPhe Met Ser 115 120 125 Phe Leu Lys Met His Tyr Met Ser Met Tyr Arg GlyLeu Gln Leu Ile 130 135 140 Lys Gly Ala Val Trp Ser Met Tyr Ser Gly GluSer Tyr Arg Glu Tyr 145 150 155 160 Leu Lys Glu Leu Glu Leu Gln Ala MetLeu Gly Glu Asn Ala Thr Leu 165 170 175 Val Ala Asn Ala Thr Gln Gly ValAsn Asn Thr Gln Thr Met Asn Asn 180 185 190 Thr Leu Leu Asn Asn Thr LysGlu Lys Asn Asn Thr Gln Arg Val Asn 195 200 205 Lys Pro Gln Glu Lys LysAla Pro Gln Lys Ala Asp Ser Pro 210 215 220 9 609 DNA Conus episcopatus9 gggagtctca acatctctgt actgcagggc caggtggtgg tggaggtgct tccagaggag 60gacagtctag aacagcccta caacatcagc atcagtgatg gccacgagtc attgattccc 120acaggggtgt tccacaaggt gtacacagtg tctgaagtgc cctcctgtta catgtacatc 180tacatggtca cggaagagac ggagttcttt gagaacctca aagagctgga acacgccctc 240aacggctccc tggatgctcc agtaccagac aagtttgcca aagatcctaa acttgatcaa 300tatatggagg tactcaaggt gaagaatgca gctccaccac cggcccctcc agcggacaga 360agtttcatac agctgtttat gagttttctg aaaatgcatt atatgtctat gtatcgtgga 420ctgcagctga taaaaggcgc cgtgtggtcc atgtactctg gggaatctta ccgagagtac 480ctgaaggaac tggagctaca ggcaatgctg ggggagaatg tcaccctggt ggcaaatgcc 540acccaagggg tgaataaaac ccagatgatg aacaacacct tgaacaacac caaaaaaaaa 600aaaaaaaaa 609 10 203 PRT Conus episcopatus 10 Gly Ser Leu Asn Ile SerVal Leu Gln Gly Gln Val Val Val Glu Val 1 5 10 15 Leu Pro Glu Glu AspSer Leu Glu Gln Pro Tyr Asn Ile Ser Ile Ser 20 25 30 Asp Gly His Glu SerLeu Ile Pro Thr Gly Val Phe His Lys Val Tyr 35 40 45 Thr Val Ser Glu ValPro Ser Cys Tyr Met Tyr Ile Tyr Met Val Thr 50 55 60 Glu Glu Thr Glu PhePhe Glu Asn Leu Lys Glu Leu Glu His Ala Leu 65 70 75 80 Asn Gly Ser LeuAsp Ala Pro Val Pro Asp Lys Phe Ala Lys Asp Pro 85 90 95 Lys Leu Asp GlnTyr Met Glu Val Leu Lys Val Lys Asn Ala Ala Pro 100 105 110 Pro Pro AlaPro Pro Ala Asp Arg Ser Phe Ile Gln Leu Phe Met Ser 115 120 125 Phe LeuLys Met His Tyr Met Ser Met Tyr Arg Gly Leu Gln Leu Ile 130 135 140 LysGly Ala Val Trp Ser Met Tyr Ser Gly Glu Ser Tyr Arg Glu Tyr 145 150 155160 Leu Lys Glu Leu Glu Leu Gln Ala Met Leu Gly Glu Asn Val Thr Leu 165170 175 Val Ala Asn Ala Thr Gln Gly Val Asn Lys Thr Gln Met Met Asn Asn180 185 190 Thr Leu Asn Asn Thr Lys Lys Lys Lys Lys Lys 195 200 11 777DNA Conus episcopatus CDS (1)..(663) 11 ggg agt ctc aac atc tct gta ctgcag ggc cag gtg gtg gtg gag gtg 48 Gly Ser Leu Asn Ile Ser Val Leu GlnGly Gln Val Val Val Glu Val 1 5 10 15 ctt cca gag gag gac agt cta gaacag ccc tac aac atc agc atc agt 96 Leu Pro Glu Glu Asp Ser Leu Glu GlnPro Tyr Asn Ile Ser Ile Ser 20 25 30 gat ggc cac gag tca ttg att ccc acaggg gtg ttc cac aag gtg tac 144 Asp Gly His Glu Ser Leu Ile Pro Thr GlyVal Phe His Lys Val Tyr 35 40 45 aca gtg tct gaa gtg ccc tcc tgt tac atgtac atc tac atg gtc acg 192 Thr Val Ser Glu Val Pro Ser Cys Tyr Met TyrIle Tyr Met Val Thr 50 55 60 gaa gag acg gag ttc ttt gag aac ctc aaa gagctg gaa cac gcc ctc 240 Glu Glu Thr Glu Phe Phe Glu Asn Leu Lys Glu LeuGlu His Ala Leu 65 70 75 80 aac ggc tcc ctg gat gct cca gta cca gac aagttt gcc aaa gat cct 288 Asn Gly Ser Leu Asp Ala Pro Val Pro Asp Lys PheAla Lys Asp Pro 85 90 95 aaa ctt gat caa tat atg gag gta ctc aaa gtg aagaat gca gct cca 336 Lys Leu Asp Gln Tyr Met Glu Val Leu Lys Val Lys AsnAla Ala Pro 100 105 110 cca ccg gcc cct cca gcg gac aga agt ttc ata cagctg ttt atg agt 384 Pro Pro Ala Pro Pro Ala Asp Arg Ser Phe Ile Gln LeuPhe Met Ser 115 120 125 ttt ctg aaa atg cat tat atg tct atg tat cgt ggactg cag ctg ata 432 Phe Leu Lys Met His Tyr Met Ser Met Tyr Arg Gly LeuGln Leu Ile 130 135 140 aaa ggc gcc gtg tgg tcc atg tac tct ggg gaa tcttac cga gag tac 480 Lys Gly Ala Val Trp Ser Met Tyr Ser Gly Glu Ser TyrArg Glu Tyr 145 150 155 160 ctg aag gaa ctg gag cta cag gca atg ctg ggggag aat gtc acc ctg 528 Leu Lys Glu Leu Glu Leu Gln Ala Met Leu Gly GluAsn Val Thr Leu 165 170 175 gtg gca aat gcc acc caa ggg gtg aat aaa acccag atg atg aac aac 576 Val Ala Asn Ala Thr Gln Gly Val Asn Lys Thr GlnMet Met Asn Asn 180 185 190 acc ttg aac aac acc aag gag aaa aac aac acccaa agg gtt aac aag 624 Thr Leu Asn Asn Thr Lys Glu Lys Asn Asn Thr GlnArg Val Asn Lys 195 200 205 ccg cag gaa aag aag gcc ccc cag aag gca gacagc ccc taacagcatc 673 Pro Gln Glu Lys Lys Ala Pro Gln Lys Ala Asp SerPro 210 215 220 tctgcagaat gagggcatca tgcctctgtt ctgcatttta aatcttcaatgtcagacacg 733 ctgtcatgag tcaggatgcc aagggttgat tctaaatgaa aaaa 777 12221 PRT Conus episcopatus 12 Gly Ser Leu Asn Ile Ser Val Leu Gln Gly GlnVal Val Val Glu Val 1 5 10 15 Leu Pro Glu Glu Asp Ser Leu Glu Gln ProTyr Asn Ile Ser Ile Ser 20 25 30 Asp Gly His Glu Ser Leu Ile Pro Thr GlyVal Phe His Lys Val Tyr 35 40 45 Thr Val Ser Glu Val Pro Ser Cys Tyr MetTyr Ile Tyr Met Val Thr 50 55 60 Glu Glu Thr Glu Phe Phe Glu Asn Leu LysGlu Leu Glu His Ala Leu 65 70 75 80 Asn Gly Ser Leu Asp Ala Pro Val ProAsp Lys Phe Ala Lys Asp Pro 85 90 95 Lys Leu Asp Gln Tyr Met Glu Val LeuLys Val Lys Asn Ala Ala Pro 100 105 110 Pro Pro Ala Pro Pro Ala Asp ArgSer Phe Ile Gln Leu Phe Met Ser 115 120 125 Phe Leu Lys Met His Tyr MetSer Met Tyr Arg Gly Leu Gln Leu Ile 130 135 140 Lys Gly Ala Val Trp SerMet Tyr Ser Gly Glu Ser Tyr Arg Glu Tyr 145 150 155 160 Leu Lys Glu LeuGlu Leu Gln Ala Met Leu Gly Glu Asn Val Thr Leu 165 170 175 Val Ala AsnAla Thr Gln Gly Val Asn Lys Thr Gln Met Met Asn Asn 180 185 190 Thr LeuAsn Asn Thr Lys Glu Lys Asn Asn Thr Gln Arg Val Asn Lys 195 200 205 ProGln Glu Lys Lys Ala Pro Gln Lys Ala Asp Ser Pro 210 215 220

What is claimed is:
 1. A synthetic nucleic acid encoding a proteinselected from the group consisting of a γ-carboxylase comprising anamino acid sequence as set forth in SEQ ID NO:2 or 4 and a proteinhaving at least 95% identity to said γ-carboxylase and capable ofγ-carboxylating Conantokin G.
 2. The synthetic nucleic acid of claim 1,wherein said nucleic acid is selected from the group consisting of anucleic acid which comprises a nucleotide sequence as set forth in SEQID NO:1 or 3 and a nucleic acid which comprises a nucleotide sequencehaving at least 95% identity to the nucleotide sequence set forth in SEQID NO:1 or
 3. 3. A vector comprising the nucleic acid of claim
 1. 4. Thevector of claim 3 which is an expression vector.
 5. A vector comprisingthe nucleic acid of claim
 2. 6. The vector of claim 5 which is anexpression vector.
 7. Host cells containing the vector of claim
 3. 8.Host cells containing the vector of claim
 4. 9. Host cells containingthe vector of claim
 5. 10. Host cells containing the vector of claim 6.11. The host cells of claim 8 which further comprises an expressionvector comprising a nucleic acid sequence encoding a conantokin.
 12. Thehost cells of claim 10 which further comprises an expression vectorcomprising a nucleic acid sequence encoding a conantokin.
 13. A methodfor producing γ-carboxylase which comprises growing the host cells ofclaim 8 under conditions suitable for growth and isolating the expressedγ-carboxylase.
 14. A method for producing γ-carboxylase which comprisesgrowing the host cells of claim 10 under conditions suitable for growthand isolating the expressed γ-carboxylase.
 15. A method for producingγ-carboxylated conantokin which comprises growing the host cells ofclaim 11 under conditions suitable for growth and isolating theγ-carboxylated conantokin.
 16. A method for producing γ-carboxylatedconantokin which comprises growing the host cells of claim 12 underconditions suitable for growth and isolating the γ-carboxylatedconantokin.
 17. An isolated γ-carboxylase selected from the groupconsisting of a γ-carboxylase comprising an amino acid sequence as setforth in SEQ ID NO:2 or 4 and a protein having at least 95% identity tosaid γ-carboxylase and capable of γ-carboxylating Cononatokin G.
 18. Amethod for producing γ-carboxylated conantokin which comprises combiningtogether a γ-carboxylase and a conantokin propeptide containing a Conusγ-carboxylase recognition sequence to produce a γ-carboxylatedconantokin and isolating the γ-carboxylated conantokin, wherein saidγ-carboxylase is selected from the group consisting of a γ-carboxylasecomprising an amino acid sequence as set forth in SEQ ID NO:2 or 4 and aprotein having at least 95% identity to said γ-carboxylase and capableof γ-carboxylating Conantokin G.